The present invention relates to hydrophobic multicomponent heparin conjugates, which are soluble in certain organic solvents but not in water, and to methods of using and making thereof. Particularly, the present invention relates to hydrophobic multicomponent heparin conjugates that are prepared by covalently binding polymeric and hydrophobic materials to heparin and that are used for treatment of patients as a antithrombogenic agent.
Thromboresistence of vascular walls requires an endothelial lining that is compatible with platelets and the plasma coagulation system. Unlike artificial vascular materials, endothelial linings do not induce serine protease activity during the coagulation process. However, when coagulability of blood is increased, the endothelium inactivates thrombin and other possible coagulation factors. Generally, the inflow site of thrombin is known to be at the surface of endothelium cells (B. Awbrey et al. J. Biol. Chem., 254, 4092–4095, 1979), and the glycosaminoglycan layer on endothelial cells is also known to have thrombin-binding sites. It is reported that if those thrombin binding sites are bound to enzymes such as serine protease, the enzymes are inactivated by antithrombin III in blood serum (C. Busch et al., J. Clin. Invest., 726–729, 1982; M. Dryjski et al., Thromb. Res., 32, 355–363, 1983).
After it was reported that an analogous structure for antithrombin binding exists in heparin molecules (J. A. Marcum et al., J. Clin. Invest., 74, 341–350, 1984; J. A Marcum et al., Biochem. Biophys. Res. Comm., 126, 365–372, 1985), methods for introducing heparin into or on the surface of solid materials to produce endothelium-like thromboresistent properties was soon developed and commercialized. Artificial surfaces, which have obtained endothelium-like thromboresistent properties by using heparin and the like can be widely used for the development of medical techniques.
Among those studies, various methods for improving blood compatibility of macromolecular surface using heparin were developed. Grode et al. synthesized heparin-silicon rubber using cyanuric chloride and radiation grafting (Grode et al., J. Biomed. Mater. Res. Symp., 3, 77–84, 1972). Merrill et al. also synthesized a conjugate wherein polyvinyl alcohol was covalently bound to heparin by glutaraldehyde cross linking (Merrill et al., J. Appl. Physiol., 29, 723–728, 1970). Eriksson and Gillerg developed a cross-linked heparin monomer in which anionic heparin was absorbed on the surface of polypropylene by heparin cross linking and use of a cationic surfactant (Eriksson and Gillerg, J. Biomed. Mater. Res., 1, 301–312, 1967).
Goosen and Sefton synthesized heparinized a styrene-butadiene-styrene elastomer (Goosen and Sefton, Thromb. Res., 20, 543–554, 1980). And, Miyura et al. designed a method of prolongation of plasma recalcification by fixing heparin on Sepharose® or polyhydroxymethacrylate (Miyura et al., J. Biomed. Mater. Res., 14, 619–630, 1980).
Jacobs et al. synthesized a covalently bonded complex of heparin and prostaglandin E1 (Jacobs et al., J. Biomed. Mater. Res., 23, 611–630, 1989). The above heparin complex was fixed on urethane by diamino-terminated polyethylene oxides, and this fixed complex was very effective for prohibiting thrombin formation. Hennink et al. developed a covalently bonded complex of heparin and human serum albumin (Hennink et al., Thromb. Res., 29, 1–13, 1983). This complex increased blood compatibility of macromolecular surfaces that were in contact with blood.
Nagaoka et al. reported the results of animal experiments that showed that a polyethylene oxide (PEO) hydrophilic spacer fixed on a macromolecular surface inhibited the attachment of platelets by absorption of blood proteins and dynamic repulsion (Nagaoka et al., Trans. Am. Soc. Artif. Intern. Organs., 10, 76—76, 1987). The inhibitory effect on platelet attachment by a spacer fixed on a heparinized surface was confirmed by Kim and Ebert (Kim and Ebert, Thromb. Res., 26, 43–57, 1982). They proved that nticoagulant activity of fixed heparin increased according to the length of spacer.
Further, Tay et al. showed that 1) a long spacer, such as PEO, made binding heparin with antithrombin III and thrombin easier; 2) heparin coupled to end groups of polyvinylalcohol was more accessible than heparin coupled to amino groups on non-terminal units of heparin macromolecules; and 3) heparin units on the surface obstructed the access of antithrombin III and thrombin (Tay et al., Biomaterials, 10, 11–15, 1989).
Schmer covalently bound heparin to agarose by using spacers such as thiophosgene or carbodiimide (Schmer, Trans. Am. Soc. Artif. Intern. Organs., 18, 321–324, 1972), and this spacer-fixed heparin had advanced binding capacity to antithrombin III.
Danishefsky and Tzeng produced heparin-agarose macromolecules by using an aminoethyl spacer (Danishefsky and Tzeng, Thromb. Res., 4, 237–246, 1974). Park et al. fixed heparin on a biomer by using a PEO spacer, and they reported that the length of such spacers should be at least 3,400 Daltons to optimize anticoagulation activity of the fixed heparin (Park et al., J. Biomed. Mater. Res., 22, 977–992, 1988). Also, heparin was fixed on the surface of spacers to optimize anticoagulation activity and to amplify surface density of the spacers. (Lin et al., J. Biomed. Mater. Res., 25, 791–795, 1991; Piao et al., Trans. Am. Soc. Artif. Intern. Organs., 38, 638–643, 1992).
Moreover, spacer systems using heparin were used for synthesis of copolymers. Triblock copolymers such as PEO-PDMS-heparin and PDMS-PEO heparin were synthesized (Grainger et al., J. Biomed. Mater. Res., 22, 231–249, 1988). These triblock copolymers coated on a segmented polyurethane surface increased nonthrombogenicity even under low flow. Vulic et al. reported a method of synthesizing polyurethane-PEO-heparin (Vulic et al., J. Polym. Sci. A, Polym. Chem., 26, 381–391, 1988), which was a modification of the solvent casting method. According to this method, coating was easy but synthesis of the heparin complex with polyurethane or polyethylene glycol was not due to problems of low efficiency and of requiring an excessive amount of solvent to suppress cross linking of heparin during the synthesis process.
To overcome the foregoing and other disadvantages, a method of making a heparin complex, comprising heparin and hydrophobic macromolecules, has been developed in which heparin is combined first with a macromolecule having multi-functional groups and then with hydrophobic materials. The result is a synthesized hydrophobic multicomponent heparin conjugate that is soluble in organic solvents but not in water. Finally, the hydrophobic multicomponent heparin conjugates of the present invention can easily be coated on the surface of all medical instruments comprising macromolecules or metals, and such coated instruments exhibit high anti-thrombosis.